A method for isolating transverse (T) - tubules from skeletal muscle has been establised by the applicant and co-workers. The isolated T-tubules vesicles retain their morphological and biochemical integrity. The specific aims and methods of this project are: a). Ca ions pump in T-tubule. 125I labeled or ferritin cross-linked Ca ions ATPase antibodies will be used to identify the Ca ions pump in the T-tubular vesicles. Active Ca ions transport into these vesicles will be measured by 45Ca ions tracing. The phosphorylation of the Ca ions pumb by gamma(32P) ATP and the nature of the phosphate bond will be determined. Also, the effect of cyclic AMP and protein kinase on CA ions transport of the T-tubules will be examined. The characteristics of the Ca ions pump in the T-tuble will be compared with those of SR. b) Transmembrane potential of the T-tubule. Previous studies demonstrated active Na ion transport across the T-tubular membrane. The generation of a membrane potential via the Na ion pump will be tested using membrane potential sensitive probes (i.e., cyanine dyes). The effect of the Ca ions pump on the T-tubular membrane potential will be studied. c). Interaction of the T-tubules and Sarcoplasmic Reticulum. The bridging proteins across the triad junctions will be extracted by KC1 and purified by Sephadex chromatography and/or isoelectric focusing. The molecular weight and its interaction with lipid bilayers will be determined. The requirement of these proteins for the reassembly of the triad junction will be determined by electron microscopy. d) Variation of ion permeabilities along th SR network. The SR network can be isolated into subfractions of longitudinal SR and light and heavy vesicles of the terminal cisternae. The passive flux rates of 22Na ion, 86Rb ion, and 36 Cl minus across the vesicles from the various regions of th SR will be compared.